Identification and Functional Analyses of Host Proteins Interacting with the P3a Protein of Brassica Yellows Virus

SIMPLE SUMMARY: A newly identified non-AUG-initiated ORF, ORF3a, encoded by members of the genus Polerovirus, is required for long-distance movement in plants. However, its functions in host protein interactions still remain unclear. Here, we systemically investigated Brassica yellows virus (BrYV)-P3a interacting proteins in plants. In total, 138 genes with annotations were obtained. Furthermore, Arabidopsis thaliana purine permease 14, glucosinolate transporter 1, and nitrate transporter 1.7 were verified to interact with P3a in vivo and were downregulated in response to BrYV during the late stages of viral infection. We used pup14, gtr1, and nrt1.7, the T-DNA insertion mutants, to preliminarily characterize their roles in the BrYV infection process. ABSTRACT: Viruses are obligate parasites that only undergo genomic replication in their host organisms. ORF3a, a newly identified non-AUG-initiated ORF encoded by members of the genus Polerovirus, is required for long-distance movement in plants. However, its interactions with host proteins still remain unclear. Here, we used Brassica yellows virus (BrYV)-P3a as bait to screen a plant split-ubiquitin-based membrane yeast two-hybrid (MYTH) cDNA library to explain the functional role of P3a in viral infections. In total, 138 genes with annotations were obtained. Bioinformatics analyses revealed that the genes from carbon fixation in photosynthetic, photosynthesis pathways, and MAPK signaling were affected. Furthermore, Arabidopsis thaliana purine permease 14 (AtPUP14), glucosinolate transporter 1 (AtGTR1), and nitrate transporter 1.7 (AtNRT1.7) were verified to interact with P3a in vivo. P3a and these three interacting proteins mainly co-localized in the cytoplasm. Expression levels of AtPUP14, AtGTR1, and AtNRT1.7 were significantly reduced in response to BrYV during the late stages of viral infection. In addition, we characterized the roles of AtPUP14, AtGTR1, and AtNRT1.7 in BrYV infection in A. thaliana using T-DNA insertion mutants, and the pup14, gtr1, and nrt1.7 mutants influenced BrYV infection to different degrees.